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Heme oxygenase-1 modulates the allo-immune response by promoting activation-induced cell death of T cells

James McDaid^*, Kenichiro Yamashita^*, Angelo Chora, Robert Öllinger^*, Terry B. Strom, Xian C. Li, Fritz H. Bach^*,1 and Miguel P. Soares^1,2

^* Immunobiology Research Center, Department of Surgery, and
Division of Immunology, Department of Medicine, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, Massachusetts, USA; and
Instituto Gulbenkian de Ciência, Oeiras, Portugal

²Correspondence: Gulbenkian Institute of Science, Inflammation laboratory, Rua da Quinta Grande, 6-2780 Oeiras, Portugal. E-mail: mpsoares{at}igc.gulbenkian.pt

SPECIFIC AIMS

Heme oxygenase-1 (HO-1), which degrades heme into three products (carbon monoxide, free iron, and biliverdin), plays a protective role in many models of disease via its anti-inflammatory, anti-apoptotic, and anti-proliferative actions. Expression of HO-1 suppresses immune responses and prolongs the survival of transplanted organs; however, the mechanism underlying these effects is not clear. We tested the hypothesis that HO-1 modulates the activation of T cells in a manner that helps sustain the survival of transplanted organs. We found that HO-1 exerts potent immunosuppressive effects in alloreactive T cells, preventing cells from mediating the rejection of transplanted organs.

PRINCIPAL FINDINGS

1. Induction of HO-1 expression by cobalt protoporphyrin IX (CoPPIX) inhibits alloantigen-driven T cell proliferation
Important as background information for the present study are our recent observations that HO-1 induction by cobalt protoporphyrin IX (CoPPIX) leads to significant prolongation of the survival of major histocompatibility (MHC) antigen mismatched (C57BL/6: H-2^b to BALB/c: H-2^d) or less extensively MHC mismatched (DBA/2: H-2^d to B6AF1: H-2^b,k/d) heart as well as pancreatic islet grafts (K. Yamashita et al., H. Wang et al., unpublished results), an observation consistent with that of others. A significant percentage of these grafts survive long term (>100 days) and recipients of such grafts become tolerant specifically to donor MHC antigens (H. Wang et al., unpublished results).

We tested the effect of systemic induction of HO-1 expression by CoPPIX in mice (BALB/c) sensitized in vivo with irradiated allogenic (C57BL/6) splenocytes. The spleen was harvested 5 days after sensitization and isolated splenocytes were restimulated in vitro with the same alloantigen (i.e., irradiated C57BL/6 splenocytes) in a mixed lymphocyte culture (MLC) assay. As expected, splenocytes from sensitized mice reexposed to the same alloantigen in vitro proliferated significantly more (P<0.005) than splenocytes isolated from naive mice (Fig. 1 ). Induction of HO-1 expression by CoPPIX resulted in significant (P<0.005) inhibition of T cell proliferation compared with untreated controls, an observation similar to that of others. Administration of zinc protoporphyrin IX (ZnPPIX), a protoporphyrin similar to CoPPIX but that does not lead to HO-1 activity, did not alter T cell proliferation vs. untreated controls (Fig. 1) . Given that CoPPIX and ZnPPIX are structurally similar and differ only in their ability to induce HO-1 activity, these data suggest that induction of HO-1 expression by CoPPIX inhibits alloantigen-mediated T cell proliferation.

View larger version (22K): [in this window] [in a new window]   Figure 1. Induction of HO-1 by CoPPIX administration suppresses alloantigen-driven T cell proliferation. BALB/c mice were either not treated or were treated for 6 days with CoPPIX or ZnPPIX (5 mg/kg/daily). All except naive mice received irradiated C57BL/6 splenocytes 2 days after the start of protoporphyrin administration. Splenocytes were harvested at day 7 and MLCs were performed using irradiated C57BL/6 splenocytes as a source of alloantigens. Data shown represent mean ±SE of 3 independent experiments, each carried out in triplicate.

2. Induction of HO-1 by CoPPIX promotes T cell apoptosis
We tested whether HO-1 may induce primed alloreactive CD4⁺ T cells to undergo apoptosis after an initial burst of proliferation, i.e., activated induced cell death (AICD). This would be consistent with the observation that HO-1 induction leads initially to a to increased proliferation of alloreactive CD4⁺ T cells (data not shown), then decreases the number of peripheral CD4 T cells (data not shown) and thus the overall ability of these cells to proliferate when rechallenged with the same alloantigens (Fig. 1) . To evaluate whether HO-1 expression induced CD4⁺ T cell apoptosis, spleen cells were treated (or not) in vitro with CoPPIX for 1 h or ZnPPIX before stimulation with an anti-CD3 antibody. Surface expression of phosphatidyl serine, an early marker of apoptosis, was assessed by flow cytometry using fluorescent-labeled annexin V. Necrosis was assessed by the vital dye 7-AAD. Necrotic cells were excluded from analysis. A higher percentage of apoptotic CD4 T cells (annexin V positive/7-AAD negative) were detected in anti-CD3-activated splenocytes treated with CoPPIX vs. ZnPPIX or untreated splenocytes (Fig. 2 ). To assess whether HO-1 expression would have similar effects in vivo, mice (BALB/c) were treated (or not) with CoPPIX or ZnPPIX, then spleen cells were harvested, labeled with 6-carboxy-succinimidyl-fluorescein ester (CSFE, a fluorescent marker allowing to trace dividing cells), and adoptively transferred into allogenic irradiated mice (C57BL/6) under the same daily protoporphyrin treatment. Splenocytes were isolated 3 days later and stained with annexin V plus an anti-CD4 antibody. There was a higher proportion of CSFE⁺CD4⁺ T cells undergoing apoptosis (annexin V positive) in CoPPIX-treated mice than in ZnPPIX-treated mice or untreated mice (data not shown). This manifested as a higher proportion of apoptotic CD4 T cells that underwent more than four cycles of proliferation (low CSFE staining) and a lower proportion in the nondividing (high CSFE staining) and one proliferation cycle population (intermediate CSFE staining). Together, these observations support the notion that HO-1 expression promotes AICD in alloreactive CD4⁺ T cells.

View larger version (29K): [in this window] [in a new window]   Figure 2. HO-1 induction by CoPPIX induces CD4 T cells to undergo apoptosis. Splenocytes were isolated from naive BALB/c mice and incubated for 1 h with normal medium or medium supplemented with CoPPIX (50 µM) or ZnPPIX (50 µM). Then T cell proliferation was induced using an anti-CD3 mAb (1 µg/mL, 72 h). Splenocytes were harvested and stained with anti-CD4 FITC + annexin-V PE (to detect apoptosis) and 7-AAD (to detect necrosis). Results shown were taken from viable (7-AAD negative) CD4+ cells. Results are representative of 3 independent experiments.

3. HO-1-induced Fas/CD95 expression mediates T cell apoptosis
We tested whether HO-1-mediated suppression of T cell proliferation was dependent on the expression of Fas/CD95, a well-established signal transduction pathway involved in AICD. Wild-type and C57BL/6-lpr Fas-deficient mice were treated with protoporphyrins as described above, splenocytes were harvested at day 6 and exposed to irradiated allogenic (BALB/c) splenocytes in an MLC assay. Contrary to wild-type mice, there was no significant suppression in MLC upon induction of HO-1 by CoPPIX in Fas-deficient animals compared with untreated or ZnPPIX-treated mice (data not shown). These data support the notion that the ability of HO-1 to induce CD4 T cells to undergo AICD is mediated via the Fas/Fas ligand signal transduction pathway.

CONCLUSIONS AND SIGNIFICANCE

Our present data uncover new actions of HO-1: HO-1 expression promotes AICD of alloantigenic CD4 T cells. This effect of HO-1 in T cells contrasts with its opposite effect in nonlymphoid cells: HO-1 appears to be proapoptotic in CD4 T cells but acts in an anti-apoptotic manner in endothelial and other nonlymphoid cells. The ability of HO-1 to promote the survival of transplanted organs is probably exerted via this dual action. That is, HO-1 acts in a cytoprotective manner in endothelial cells to prevent T cell-mediated injury in the graft endothelium while inducing AICD of those T cells that mediated cytotoxicity, and thus promotes graft survival.

View larger version (34K): [in this window] [in a new window]   Figure 3. Expression of HO-1 induces alloreactive CD4 T cells to undergo AICD, thus promoting graft survival. It has been clearly established that HO-1 expression promotes the survival of transplanted organs. This protective effect has been associated primarily with the ability of HO-1 to act in an anti-inflammatory and cytoprotective manner in the graft vasculature. The results illustrated in this paper suggest that HO-1 also acts in CD4 T cells to promote AICD via generation of one or a combination of several products of HO-1 action on heme (e.g., carbon monoxide, biliverdin, and/or bilirubin. The possibility that modulation of the free pool of cellular iron may be implicated in these effects cannot be excluded. Whether these molecules act directly on CD4 T cells or via their action on antigen-presenting cells remains to be established. Our data support the notion that the ability of HO-1 to promote CD4 T cell AICD is mediated via the Fas/FasL signal transduction pathway.

FOOTNOTES

To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.04-2217fje; doi: 10.1096/fj.04-2217fje

¹ M.P.S. and F.H.B. contributed equally to this work.

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